The invention relates to acid-labile and enzymatically cleavable compounds for in-vivo and in-vitro diagnosis with near-infrared radiation (NIR radiation), the use of these compounds as optical diagnostic agents and therapeutic agents and diagnostic agents that contain these compounds.
Near-infrared imaging is a non-invasive diagnostic process, in which the high permeability of biological tissue to light on a wavelength 650-1000 nm is exploited. In contrast to the light of the ultraviolet and visible spectral range, which can penetrate only into the topmost millimeter of the tissue, penetration depths into the tissue of up to several centimeters is achieved with use of near-infrared light. The reasons for the basically small penetration depth of light are the absorption of endogenous dyes, mainly hemoglobin and water, which in the spectral range of the near-infrared light, however, have minimum values of between 650 and 1000 nm. This spectral range of the maximum optical tissue transparency is therefore also named a diagnostic/therapeutic window (Boulnois, J., Lasers Med Sci 1986, 1:47-66).
In addition to the modern imaging processes, such as diagnostic radiology, magnetic resonance tomography or ultrasonic diagnosis, another process for graphic tissue visualization is thus available to the diagnostician (Haller, E. B., Time-Resolved Transillumination and Optical Tomography. J. Biomed Optics 1996, 1:7-17).
The use of NIR radiation for site-dependent recording of blood flow and degree of oxygenation in the brains of babies by the detection of the absorption of hemoglobin/deoxyhemoglobin is a process that has been known and used for years (Jxc3x6bsis, F. F., Science 1977, 198: 1264-67; Chance, B.; Leigh, J. S.; Miyake, H. et al., Proc Natl Acad Sci USA 1988, 85: 4971-75; Benaron, D. A. et al., Science 1993, 33: 369A.).
The basic problem when near-infrared radiation is used is the strong scattering of light, so that even in the case of different photophysical properties, this object is poorly distinguished from an object with sharp edges and its surrounding area. The problem increases with increasing removal of the object from the surface and can be considered as a main limiting factor both in the case of transillumination and in the detection of fluorescence radiation. As contrast media, dyes, which mark the optical properties of the tissue and result in an increased absorption and fluorescence of the tissue that is to be detected, can therefore make unambiguous detection possible even with poor site resolution. In this case, the absorption behavior of such dye compounds can be used as imaging information. If the dyes, moreover, have the property of emitting the absorbed energy as fluorescence radiation, the latter can also be used as imaging information. In this case, the fluorescence radiation that is red-shifted relative to the excitation radiation is detected separately. The advantage exists, i.a., in that the tissue itself has an extremely low inherent fluorescence in the NIR range and thus the background is minimal. (S. Folli et al., Cancer Research 54, 2643-9 (1994); B. Ballou et al., Cancer Immunol. Immunother. 41, 257-63 (1995); X. Li et al., SPIE Vol. 2389, 789-98 (1995)).
In fluorescence diagnosis, the precondition in this respect is to detect an adequate difference that is as great as possible in the fluorescence emission between the tissue that is to be detected and the surrounding tissue. This can be achieved in principle by a difference in the concentration of the fluorescence dye at a certain time after the substance administration has been achieved. In particular for diagnosis in deeper tissue layers, this difference in the use of substances with unspecific concentration behavior is often inadequate.
The object of the invention is to make available new compounds that overcome the drawbacks of the prior art.
The object is achieved according to the invention by compounds of general formula (I)
(Fxe2x80x94L)mxe2x80x94Axe2x80x83xe2x80x83(I),
in which
F stands for a dye molecule with at least one absorption maximum of between 600 and 1200 nm,
L stands for a linker structure, which contains an acid-labile and/or enzymatically cleavable bond,
m is a number between 1 and 80,
whereby if m is a number between 1 and 3,
A represents a dye molecule with at least one absorption maximum of between 600 and 1200 nm, an antibiotically or anticytostatically active molecule, a biomolecule, a non-biological macromolecule or a compound Bxe2x80x94(Lxe2x80x94W)o or Dxe2x80x94(Lxe2x80x94W)o, whereby
D is a non-biological macromolecule,
B is a biomolecule,
L has the above-mentioned meaning,
W represents an antibiotically or anticytostatically active molecule,
o is a number between 1 and 20,
and whereby if m is a number between 4 and 80,
A represents a biomolecule, a non-biological macromolecule or a compound Bxe2x80x94(Lxe2x80x94W)o or Dxe2x80x94(Lxe2x80x94W)o, whereby
D, B, L, W and o have the above-mentioned meanings.
The special property with respect to the in-vivo detection of the near-infrared fluorescence emission of the compounds according to the invention consists in the fact that the latter have little or even no fluorescence emission, and an increase of the fluorescence signal occurs only after this construct is cleaved or after the dye is cleaved off from the construct on the target site (e.g., tumors, inflammations). The effective difference of the fluorescence signal between the tissue that is to be detected and the surrounding tissue is consequently marked by the fact of
a) the concentration difference based on pharmacokinetics mechanisms and
b) by the difference in the fluorescence quantum yield at the time of the diagnosis.
It has been found that the fluorescence of the dyes is quenched when a dye molecule is coupled to another molecule (dimer) while obtaining the compounds according to the invention, i.e., an extremely low fluorescence emission occurs in comparison to the corresponding dye molecule in the unbonded state. It has been found, moreover, that a comparable quenching occurs when other molecules with aromatic structures, which can be both dyes and active ingredients (e.g., cytostatic agents or antibiotic agents), are coupled with the fluorescence dye. Surprisingly enough, a quenching also occurs when the dyes are coupled to the antibodies, antibody fragments and proteins.
In principle, the dyes, which are structural components of the compounds according to the invention, must be distinguished in their monomeric unconjugated form by high molar absorption coefficients and high fluorescence quantum yields.
Preferred compounds of general formula I according to the invention are distinguished in that F and/or A stand for a polymethine dye, tetrapyrrole dye, tetraazapyrrole dye, xanthine dye, phenoxazine dye or phenothiazine dye.
Especially preferred are the structures from the class of polymethine dyes, since the latter have absorption maxima with very high molar absorption coefficients in the near-infrared spectral range of between 700 and 1000 nm ("xgr" up to 300,000 1 molxe2x88x921 cmxe2x88x921), such as, for example, cyanine dyes, squarilium dyes and croconium dyes, as well as merocyanine and oxonol dyes.
Those compounds of general formula (I) according to the invention are also preferred in which F and/or A stand for a cyanine dye of general formula II 
in which
R1 to R4 and R7 to R10, independently of one another, stand for a fluorine, chlorine, bromine, iodine atom or a nitro group or for a radical xe2x80x94COOE1, xe2x80x94CONE1E2, xe2x80x94NHCOE1, xe2x80x94NHCONHE1, xe2x80x94NE1E2, xe2x80x94OE1, xe2x80x94OSO3E1, xe2x80x94SO3E1, xe2x80x94SO2NHE1, xe2x80x94E1, whereby E1 and E2, independently of one another, stand for a hydrogen atom, a saturated or unsaturated, branched or straight-chain C1-C50 alkyl chain, whereby the chain or parts of this chain optionally can form one or more aromatic or saturated cyclic C5-C6 units or bicyclic C10 units, and whereby the C1-C50 alkyl chain is interrupted by 0 to 15 oxygen atoms and/or 0 to 3 carbonyl groups and/or is substituted with 0 to 5 hydroxy groups, 0 to 5 ester groups, 0 to 3 carbon groups, 0 to 3 amino groups, and whereby in each case adjacent radicals R1-R4 and/or R7-R10 can be linked with one another with the formation of a six-membered aromatic carbon ring,
R5 and R6, independently of one another, stand for a radical xe2x80x94E1 with the above-indicated meaning or for a C1-C4 sulfoalkyl chain,
and/or R1 to R10 stand for a linkage with L,
Q is a fragment 
xe2x80x83in which
R11 stands for a hydrogen, fluorine, chlorine, bromine or iodine atom or a nitro group or a radical xe2x80x94NE1E2, xe2x80x94OE1 or xe2x80x94E1, whereby E1 and E2 have the above-indicated meaning or R11 stands for a linkage with L,
R12 stands for a hydrogen atom or a radical E1 with the above-indicated meaning,
b means a number 0, 2 or 3,
X and Y, independently of one another, represent O, S, xe2x80x94CHxe2x95x90CHxe2x80x94 or a fragment 
xe2x80x83in which
R13 and R14, independently of one another, stand for hydrogen a saturated or unsaturated, branched or straight-chain C1-C10 alkyl chain, which can be interrupted by up to 5 oxygen atoms and/or substituted with up to 5 hydroxy groups, and whereby radicals R13 and R14 can be linked with one another while forming a 5- or 6-membered ring.
Another subject of the invention are compounds of general formula (I), in which dyes with a therapeutically active molecule are linked via a physiologically cleavable bond, or dyes and active ingredients are coupled via physiologically cleavable bonds to biomolecules or non-biological carrier molecules.
Especially preferred are constructs, in which the fluorescence of the dye in the coupled state is quenched, and the therapeutic activity of the active molecule is masked by the coupling to the dye or carrier molecule (pro-drug effect). The cleavage of the bond results in an increase of fluorescence emission with simultaneous release of the activity of the active ingredient.
Active ingredients W and/or A in general formula (I) according to the invention are, for example, the compounds that are cited below:
Antibiotics: aclacinomycin, actinomycin F1, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, doxorubicin, epirubicin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, plicamycin, porfiromycin, puromycin, streptonigrin, tubercidin, zorubicin;
Folic acid analogs: denopterin, metothrexate, pteropterin, trimetrexate;
Pyridimidine analogs: ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil;
Purine analogs: fludarabine, 6-mercaptopurine, thiamiprine, thioguanine and derivatives of the above-mentioned compounds,
Alkylating substances: alkylsulfonates, aziridines, ethylenimines, methylmelamines, nitroureas, nitrogen mustard compounds;
Hormonally active substances such as androgens, antiadrenals, antiandrogens, antiestrogens, estrogens, LH-RH analogs and progestogens,
as well as other cytostatically active substances, such as taxol and taxol derivatives.
Other active ingredients are photodynamically active substances, which are distinguished by the capability, after excitation, to exert a photosensitizing action by forming cytotoxic singlet oxygen and radicals. Such compounds are primarily tetrapyrroles or tetraazapyrroles, for example porphyrins, benzoporphyrins, chlorines, purpurines, phthalocyanines, naphthalocyanines and derivatives of the above-mentioned compounds. Other compounds are expanded porphyrins, porphycenes and oxazine or phenoxazine dyes.
The chemical bond, which is contained in linker structure L according to general formula (I), is structurally constituted in such a way that the latter is cleaved in the case of certain physiological parameters that characterize diseased tissues (tumors) and that are distinguished from normal tissue areas.
It is described in the literature that tumors are characterized by low pH in comparison to the normal tissue. While the intracellular pH is largely identical (about pH 7.4), the extracellular pH in tumors is reduced to up to 0.5 pH units. Also, inflammations, especially of the bacterial type, are characterized by reduced pH. The methods for determining the pH are, i.a., measurements with microelectrodes, fluorescence measurements with pH-sensitive fluorescence samples and measurements with MR probes (R. J. Gillies et al., Am. J. Physiol. 267, pC 195-203 (1994),
G. R. Martin and R. K. Jain, Microvascular Research 46, 216-230 (1993),
L. E. Gerweck and K. Seetharaman, Cancer Research 56, 1194-1198 (1996)),
K. Engin et al., Int. J. Hyperthermia 11 (1995) 211-216,
K. Engin et al., Int. J. Radiation Oncology Biol. Phys. 29 (1994) 125-132,
G. Helmlinger et al., Nature Medicine 3 (1997) 177-182.
Another subject of the invention are therefore compounds with linker structures L, which are cleaved by reduced physiological pH values. Such structures are, for example, alkylhydrazones, acylhydrazones, arylhydrazones, sulfonylhydrazones, imines, oximes, acetals, ketals, orthoesters corresponding to the fragments 
in which p stands for a number between 2 and 4.
In addition to the cleavage that is based on reduced pH, the cleavage of the compounds according to the invention can also be carried out by enzymes, which are present in increased concentration in the tissues that are to be detected (e.g., tumors, bacterial inflammations).
Another subject of the invention are therefore compounds with linker structures L, which can be cleaved enzymatically. Enzymatically cleavable linker structures are, for example, those that are cleaved by cathepsins, peptidases, carboxypeptidases, xcex1-and xcex2-glucosidases, lipasesi oxidases, phospholipases, phosphatases, phosphodiesterases, proteases, elastases, sulfatases, reductases, transferases and bacterial enzymes, for example penicillin-amidases as well as xcex2-lactamases (P. D. Senter et al., Bioconjugates Chem. 6 (1995), 389-94).
Preferred enzymatically cleavable structures are short-chain peptide sequences, such as, for example, sequences that contain the amino acid sequence Val-Leu-Lys.
The kinetics that results in a concentration in the tissue that is to be detected or in a corresponding concentration gradient at a certain time after administration must correlate both with the kinetics of the cleavage of the compounds according to the invention and with the kinetics of the removal of the released dye molecule and result in a synergistic effect.
Other preferred compounds of general formula (I) according to the invention are distinguished in that A and/or B stands for an antibody, its conjugates and fragments, specific peptides and proteins, receptors, enzymes, enzyme substrates, nucleotides, natural or synthetic ribonucleic acids or deoxyribonucleic acids or their chemical modifications, such as aptamers or antisense oligonucleotides, lipoproteins, lectins, carbohydrates, mono-, di- or trisaccharides, linear or branched oligosaccharides or polysaccharides or -saccharide derivatives or for a dextran.
Also, the compounds of general formula (I) according to the invention are preferred in which D represents polyethylene glycol, polypropylene glycol, polylysine or polylysine dendrimers or derivatives thereof.
The linkage of structural elements A, D, B, L and W is carried out either directly or via commonly used functional groups. Such groups are, for example, esters, ethers, secondary and tertiary amines, amides, thiourea, urea, carbamate groups or maleimido structures.
Another subject of the invention is the use of the compounds of general formula I according to the invention for in-vivo diagnosis of diseased tissue areas with use of NIR radiation and for treatment of diseased tissue areas.
The subject of the invention is also an optical diagnostic agent for in-vivo diagnosis of diseased tissue areas with use of NIR radiation, which contains at least one compound of general formula (I) according to the invention.
These agents are produced according to the methods that are known to one skilled in the art, optionally with use of commonly used adjuvants and/or vehicles as well as diluents, etc. These include physiologically compatible electrolytes, buffers, detergents and substances for matching osmolarity as well as for improving stability and solubility. The measures that are commonly used in pharmaceutics ensure the sterility of the preparations during production and especially before administration.
The synthesis of dyes F and A is carried out according to methods that are known in the literature, e.g.
F. M. Hamer in The Cyanine Dyes and Related Compounds, John Wiley and Sons, New York, 1964;
J. Fabian et al., Chem. Rev. 92 (1992) 1197;
L. A. Ernst et al., Cytometrie [Cytometry] 10 (1989) 3-10;
P. L. Southwick et al., Cytometrie 11 (1990) 418-430;
R. B. Mujumdar et al., Bioconjugate Chem. 4 (1993) 105-11;
E. Terpetschnig et al., Anal. Biochem. 217 (1994) 197-204;
J. S. Lindsey et al., Tetrahedron 45 (1989) 4845-66, EP-0591820 A1;
L. Strekowski et al., J. Heterocycl. Chem. 33 (1996) 1685-1688;
S. R. Mujumdar et al., Bioconjugate Chem. 7 (1996) 356-362;
M. Lipowska et al., Synth. Commun. 23 (1993) 3087-94;
E. Terpetschnig et al., Anal. Chim. Acta 282 (1993) 633-641;
M. Matsuoka and T. Kitao, Dyes Pigm. 10 (1988) 13-22, and
N. Narayanan and G. Patronay, I. Org. Chem. 60 (1995) 2361-95.
The dyes are synthesized in a way similar to methods that are known in the literature with substituents that contain acid-labile or enzymatically cleavable bonds or from which such bonds are produced after coupling; e.g., according to
B. M. Mueller et al., Bioconjugate Chem. 1 (1990) 325-330;
K. Srinivasachar and D. M. Neville, Biochemistry 28 (1989) 2501-09;
D. M. Neville et al., J. Biol. Chem. 264 (1989) 14653-61;
T. Kaneko et al., Bioconjugate Chem. 2 (1991), 133-41;
B. A. Froesch et al., Cancer Immunol. Immunother. 42 (1996), 55-63 and
J. V. Crivello et al., J. Polymer Sci: Part A: Polymer Chem. 34 (1996) 3091-3102.